The definition of neoplasia noted in Chapter 2 was developed from observations of neoplastic disease in vivo. The criteria of transformation in cell culture listed in Table 14.1 did not define neoplastic disease in vitro but rather described a number of its characteristics. While neoplasia in vivo and cell transformation in vitro exhibit many apparent dissimilarities, considerable effort has been expended in trying to identify analogies in the natural history of the development of the neoplastic process in vivo and in vitro. We have already noted the changes spontaneously occur- ring in SHE cells transformed with chemical carcinogens or ionizing radiation (Table 14.2). However, a number of studies have been directed toward more controlled investigations of po- tential “stages” occurring during the development of cell transformation in vitro.
As discussed earlier (Chapter 7), there is substantial evidence that the stage of initiation requires that division of the cell susceptible to the carcinogenic agent occur while the carcinogenic agent is present. This phenomenon was perhaps even more definitively demonstrated in cell culture with chemical carcinogenesis of SHE cells (Berwald and Sachs, 1963). These authors demon- strated that, unless the carcinogen remained in the culture medium for a period at least equiva- lent to that of a single cell cycle of the cells being treated, no transformation would occur. This finding led to the demonstration in cell culture that at least one round of cell division was neces- sary for the neoplastic transformation to be “fixed” (cf. Kakunaga, 1975). A similar phenome- non held true for both DNA tumor virus- and x-irradiation–induced transformation in cell culture, although the latter effect appeared to require two cell generations (Borek and Sachs,1968). Sachs (1974) had suggested that possibly two cell divisions are required to fix transfor- mation by chemicals in cell culture as well. In the C3H/10T1/2 cell line, “transformation” is most efficient when the carcinogen is applied just prior to the onset of DNA synthesis in cultures synchronized for this parameter (Jones et al., 1977; Grisham et al., 1980).
In several instances, transformation induced by known carcinogens (such as polycyclic hydrocarbons) indicated that this process in vitro follows the kinetics of a single-hit phenome- non, exhibiting no threshold (cf. DiPaolo et al., 1971a), that is, lacking a “no-effect” level of the carcinogenic agent (Chapter 13). However, in the C3H10T1/2 cell line, the frequency of initia- tion or formation of foci by chemical carcinogens was found to decrease with increasing cell density of the culture (Huband et al., 1985). Furthermore, polycyclic hydrocarbons (such as benzo[a]pyrene) are themselves capable of inducing quiescent, confluent cultures of hamster embryo cells to enter DNA synthesis, after which cell transformation becomes apparent (Mironescu and Love, 1974).
The first relatively clear demonstration of distinct stages in the transformation of cells in culture was the report by Mondal and Heidelberger (1976) of cells of the C3H/10T1/2 mouse line that were initiated with ultraviolet radiation and promoted with TPA (Table 14.3). Later studies by Sanchez et al. (1986), using N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) as an initiating agent for the C3H10T1/2 cell line followed by promotion with TPA, induced numerous foci of morphologically transformed cells. Removal of TPA from the medium of such dishes containing foci resulted in regression of up to 84% of the foci and loss of morphological transformation. Stages in the induction of transformation in this cell line by chemicals (Lillehaug and Djurhuus,
1982) and ionizing radiation (Han and Elkind, 1982) have also been reported (Table 14.4). In addition, apparent steps in the transformation of other cell lines have also been identified (Tho- massen and DeMars, 1982; Table 14.4). The JB6 mouse epidermal cell line (Colburn et al.,1982) has been used to identify genes potentially involved in the stage of tumor promotion in vitro as well as more broadly (Lerman et al., 1987). Transformed foci may be induced in this cell line by several different promoting agents.
In primary cultures, the natural history of neoplastic development has been best defined in the hamster embryo cell transformation system (Barrett and Ts’o, 1978). After initiation by either chemicals (Poiley et al., 1979) or radiation (DiPaolo et al., 1981), a clear promoting effect by TPA could be demonstrated in hamster embryo cells. Poiley et al. (1979) demonstrated that TPA could act as either an inhibitor or a promoting agent, depending on the length of time be- tween initiation and the first addition of the promoting agent. Furthermore, Rivedal and Sanner (1982) demonstrated that reversal of the timing of the initiation and promotion stages resulted in no significant enhancement of transformation frequency in this system. Using this same system, Rivedal and Sanner (1981) demonstrated that several metal salts—including nickel sulfate, cad- mium acetate, and potassium chromate—could act to promote the transformation of hamster
Table 14.3 Transformation of C3H/10T1/2 Cells by Ultraviolet Light and Tetradecanoylphorbol Acetate (TPA)a
embryo cells initiated by benzo[a]pyrene. That spontaneously initiated cells may occur in these cultures was reported by Nakano and Ts’o (1981), who found subpopulations of cells lacking contact inhibition of cell division and anchorage dependence for growth in cell cultures established from hamster embryos. Sodium fluoride acts as a promoter in this system but was also shown to induce transformation in SHE cells in the absence of other initiating agents (Tsutsui et al., 1984). Two other promoting agents, mezerein (Tu et al., 1992) and diethylstil- bestrol (Hayashi et al., 1996), can induce transformation in cultured SHE cells as well. As pointed out by Boyd and Barrett (1990), these agents induce DNA damage, principally clasto-genic effects suggesting that SHE cells are either quite sensitive to progressor agents or do in fact have a significant population of spontaneously initiated cells. In fact, Ueo et al. (1990) found that early-passage, normal diploid SHE cells contained a transient subpopulation of cells lacking density-dependent inhibition of cell division and presumably capable of forming mor- phologically transformed colonies. The interesting effect of lowered pH as a promoting agent in this system described by LeBoeuf and colleagues (cf. Kerckaert et al., 1996) may be explained by a concomitant increase in cell division caused by such lowered pH. Interestingly, the non–phorbol ester tumor promoter, okadaic acid, does not promote morphological transforma- tion in SHE cells, although it is quite effective in mouse epidermis in vivo (Rivedal et al., 1990). In addition to the other examples of initiation/promotion systems in primary cultures, stages in the neoplastic development of other cell types in primary culture have been reported for the mouse submandibular gland epithelium (Wigley, 1983), rat embryo cells (Chouroulinkov and Lasne, 1978), and mouse epidermal cells (Yuspa et al., 1981). When hepatocytes were initiated in vivo and continued on several carcinogens for 6 to 12 weeks, TPA did not stimulate DNA synthesis or promote growth of primary cultures obtained from such livers (Kayano et al., 1982). In contrast, studies by Kaufmann et al. (1988) demonstrated that phenobarbital induced a prolif- eration of foci in primary cell culture from animals treated with methyl(acetoxymethyl)nitro- samine and then explanted to primary culture. These authors suggested that the proliferation of putatively initiated hepatocytes required the presence of the promoting agent as shown in vivo from other studies (Chapter 7). Kopelovich (1981) has shown that fibroblasts cultured from indi- viduals genetically predisposed to colorectal cancer are less sensitive to the toxic effects of the promoting agent TPA, and Friedman et al. (1984) demonstrated that in cultures of colonic epi- thelial cells, TPA stimulated DNA synthesis in cells derived from patients with familial polypo- sis but not in control individuals. Later studies (Antecol, 1988) did not appear to substantiate these findings.
From all of these studies it becomes apparent that transformation of cells in culture may exhibit identifiable stages of initiation and promotion, with a number of the characteristics seen in these stages in vivo (cf. Boyd and Barrett, 1990). Some of the differences in vivo versus in vitro may be related either to the genetic constitution of the cells under study, e.g., cell lines, or the heterogeneity of the primary cultures utilized, e.g., SHE cells. In either event, the similarities are much greater than the differences, arguing that studies of the multistage nature of neoplastic development as seen in cell culture may have significant application to an understanding of mechanisms occurring both in vivo and in vitro in these individual stages. Because of the ability to control the cellular environment in vitro, cell culture may offer significant advantages as long as the investigator maintains a close association of findings in vitro with those in vivo.